PROJECT ABSTRACT Epigenetic factors have emerged as crucial players in metabolic disorders and in aging, both of which are typically associated with a wide range of gene expression changes. The overall objective of this proposal is to investigate the roles of a novel histone modification, lysine malonylation, and its regulation by the NAD+- dependent protein deacylase SIRT5, as a novel histone modifier in epigenetic regulation of gene expression in response to metabolic changes. The model is based on our recent identification of histone H2B lysine 5 (H2BK5) as a site of malonylation regulated by SIRT5. We propose that the dynamic malonylation of histone H2B by cellular malonyl-CoA and demalonylation by SIRT5 regulates chromatin structure and gene transcription. Two specific aims are developed to test this model globally in mouse liver and then mechanistically in cultured cells. First, genomic regions bound with malonylated histones (H2BK5) and SIRT5 will be compared using ChIP-seq using tissues from wild type and Sirt5-/- mice. These studies will aim to identify the sites of SIRT5-mediated histone demalonylation. The functional consequences of histone malonylation will be evaluated by comparing gene transcriptional changes using RNA-seq between wild type and Sirt5-/- mice. This data set will be compared to the sites where SIRT5 binds to the genome and demalonylates histones to identify the direct genomic sites of SIRT5 action. Second, we will study in mechanistic details how histone malonylation is dynamically regulated by SIRT5 and by fluctuations in cellular malonyl-CoA that occur during feeding and fasting in mice. In primary cultured mouse hepatocytes, we will also test the effect of feeding malonate (which is converted into malonyl-CoA intracellularly) and the effect of manipulating the cellular synthesis or degradation of malonyl-CoA via acetyl-CoA carboxylase (synthesis) and malonyl-coA decarboxylase (degradation). This research proposal takes the first step towards understanding the function of this newly discovered histone modification, malonylation, and its eraser, SIRT5, in epigenetic regulation. It will contribute significantly to our longstanding effort of unveiling the ?histone code?, its intersection with intermediary metabolism, and to advance our knowledge of gene expression regulation by epigenetic factors. Results from the proposed project will therefore permit mechanistic follow-up studies of the significance of histone malonylation and SIRT5 in epigenetic regulation and how dysregulation may contribute to both pathological conditions, such as metabolic syndrome and diabetes, and to normal aging.